Cancer progression has been more and more associated to new genetic alterations or dedifferentiation processes.
More specifically, in prostate cancer, prostate cancer cells initially require androgens to proliferate and the progression of the disease is stopped by hormone therapies that decrease androgen signaling. Unfortunately, in many cases, after variable periods of time, prostate cancer becomes resistant to hormone therapies, entering the stage of “castration-resistant prostate cancer” or “hormone-refractory prostate cancer” and starts progressing again.
This renewed progression of prostate cancer is due to the selection over time of a population of cancer cells that can proliferate with low concentrations of androgens or in the total absence of them. This hormone resistance is mediated by two types of mechanisms:
(1) Androgen-dependent resistance, in which cancer cells are still dependent from androgen signaling but develop: (a) a hyperactivity of the androgen receptors (AR) that now need little or no androgens to be activated and/or (b) an ectopic production of androgens that are now synthesized autonomously by cancer cells which are insensitive to hormone suppressive therapies.
(2) Androgen-independent resistance, in which cancer cells become completely independent from androgen signaling because of (a) a profound de-differentiation of the cell, often accompanied by epithelial-mesenchymal transition (EMT), which increases malignancy and confer the ability to proliferate without androgens; and/or (b) the acquisition of an intrinsic resistance to apoptosis mediated by the over-expression of anti-apoptotic proteins, which counteract the cell death normally induced by the absence of androgens.
Clearly, efficient treatments of hormone-refractory prostate cancer need to target both androgen-dependent and androgen-independent mechanisms of resistance.
Several pharmaceutical companies are developing a second generation of hormone therapies that try to overcome androgen-dependent mechanisms of resistance either using better antagonists of the androgen receptors (MDV3100, Medivation) or by blocking the ectopic production of androgens by cancer cells (abiraterone acetate, J&J; and TAK 700, Takeda) and also by targeting both mechanisms (TOK-001, Tokai Pharmaceuticals).
However, there is currently no efficient treatment of androgen-independent hormone-refractory prostate cancer. In addition, it may be difficult or costly to determine if a hormone-refractory prostate cancer is based on an androgen-dependent or an androgen-independent mechanism of resistance. There is thus a need for new treatments able to target both androgen-dependent and androgen-independent hormone-refractory prostate cancer. Similarly, there is also a need for treatments able to target other progressing cancers characterized by cell de-differentiation.
LINE-1 (L1) is a mammalian family of retrotransposable elements, i.e. mobile DNA sequences able to relocalize throughout the genome via RNA intermediates that are reverse-transcribed and inserted at new genomic locations. The canonical, full-length L1 element is ˜6 kilobases (kb) in length and has two open reading frames, ORF1 and ORF2 coding for two proteins. ORF1 codes for 40 kDa RNA binding protein. ORF2 codes for a 149 kDa protein which is a reverse transcriptase (RT) that also has an endonuclease domain. This molecular machinery makes L1 elements among the few autonomous transposable elements in the human genome and the more abundant by mass. Each cell contains approximatively 500,000 copies of L1 accounting for 17% of the human genome. Most of L1 copies are truncated but it is calculated that there are approximatively 100 copies of full length L1 elements that encode for functional proteins.
The ORF2-produced L1-reverse transcriptase protein (L1-RT) has been increasingly involved in the pathophysiology of cancer. In normal adult tissues, L1-RT activity is strongly repressed by methylation of its promoter (Schulz, 2006). In contrast, L1-RT activity is strongly activated in several types of cancer.
Hypomethylation of LINE-1 is the main candidate mechanism for the pathological reactivation of this retrotransposon during cancer progression, and has been reported in several human malignant neoplasiae, such as colon (Estecio, 2007), liver (Tangkijvanich, 2007), prostate (Cho, 2007), bladder (Neuhausen, 2006), ovary (Pattamadilok, 2008), leukemia (Roman-Gomez, 2005). In addition, the degree of LINE-1 hypomethylation has been reported to be correlated with tumor progression and prognosis in several types of cancer; ovary (Pattamadilok, 2008), prostate (Cho, 2007), liver (Tangkijvanich, 2007). The potential influence of LINE-1 on cancer is strengthened by direct functional tests that have evidenced a strong correlation between L1-RT activity and the proliferation-transformation status of various cancer cell lines in culture (Mangiacasale 2003). Finally, an increase in the activity of L1-RT in human epithelial cancers has been recently demonstrated measuring L1-RT dependent somatic retrotransposition (Lee, 2012).
In addition to hypomethylation, a supplementary more specific mechanism seems to mediate the overexpression of L1-RT in hormone-refractory prostate cancer cells. Thus, in poorly differentiated hormone-refractory prostate cancer cells, it has been shown that the over expression of L1-RT is due to the low expression of the proteins PIWIs. PIWIs proteins are highly expressed in differentiated prostate cells and inhibit the transcription of LINE-1 (Lin, 2009).
The reactivation of LINE-1 in cancer cells is known to cause retrotransposition events which, in turn, produce dramatic consequences like mutations and DNA double strand breaks, a series of events that rapidly leads to global genetic instability (Gilbert, 2005; Gasior, 2007). In addition to genetic instability, L1-proteins seem also able to control gene expression epigenetically (for review see Sinibaldi-Vallebona, 2006).
L1-proteins can induce genetic instability by several mechanisms. The most evident is insertional mutagenesis, a process through which the insertion of a retroelement within a protein-coding gene can be equivalent to a functional gene “knock-out” (reviewed by Kazazian, 1998), or, on the contrary, give rise to a new coding unit (Nekrutenko, 2001). Retrotransposons can also act as cis-acting transcriptional regulatory sequences, providing DNA binding sites for a variety of Pol III- and Pol II-associated transcription factors (reviewed by Tomilin, 1999), and hormone—(Norris, 1995; Babich, 1999) and retinoic acid—(Islam, 1993) receptor-dependent transcriptional enhancers. Retrotransposition can also influence posttranscriptional regulation by providing novel splicing sites in coding genes (Ashworth, 1990; Feuchter-Murthy, 1993) or by insertion into specific introns it can considerably reduce the transcriptional elongation of the targeted gene (Han, 2004). Furthermore, LINE-1 sequences can provide new promoters in the sense (Ferrigno, 2001) or antisense (Speek, 2001) orientation, thus triggering the activation of existing genes and modulating tissue-specific expression. Finally, L1-RT mediated somatic retrotransposition has been recently proposed as a selection mechanism increasing the presence of the most aggressive cancer clones in epithelial cancers (Lee, 2012).
In addition to genetic instability, the activity of L1-RT seems also able to influence epigenetically the cell fate providing phenotypic variations in mammals (for a review, see Whitelaw, 2001). Although not all molecular steps are fully understood, the epigenetic control exerted by L1-RT has been recently shown by the profound reprogramming of the transcriptome, induced in tumor cell by the treatment with pharmacological inhibitors of L1-RT. This reprogramming involves both protein-coding and non-coding sequences associated with a global reorganization of the nuclear chromatin conformation, Further studies now suggest that L1-RT participates to a tumor-protecting mechanism by profoundly altering the miRNA-mediated regulation of transcription. L1-RT seems able to inactivate double stranded miRNA primary transcripts by shifting the equilibrium towards RNA/DNA hybrid structures via reverse transcription.
Recently the over-expression of L1-ORF2 protein, which contains the RT domain, has been linked to specific pathophysiological processes involved in prostate cancer progression and malignancy. In prostate cancer, the activation of the AR (Androgen Receptor) induces DNA recombination at specific site in the genome (Lin, 2009). These events involve translocation of the 5′ untranslated region of the AR target gene TMPRSS2 to two members of the ETS family of genes, ERG and ETV1 (Tomlins, 2005). These rearrangements lead to androgen dependent over-expression of ETS transcription factors, most frequently the proto-oncogenes ETV1 or ERG, which have been proposed as key factors in stimulating the development and aggressiveness of prostate cancers (Shaffer, 2006). This site specific AR-induced translocation event needs the recruitment of L1-ORF2 protein. Thus, over expression of L1-RT proteins greatly enhances AR-induced site specific translocation of TMPRSS2, while competition of the active proteins with an inactive mutant greatly reduces it.
Supporting a causal relationship between LINE-1 activity and tumor progression, suppression by RNA interference of L1-RT in a human melanoma cell line (A375) results in a strong inhibition of cell proliferation in vitro (pure cytostatic effect) and tumor growth in vivo in xenografted mice (Sciamanna, 2005; WO2006069812A2).
An increasing body of compelling evidences thus suggests that the transcriptional reactivation of LINE-1 expression—notably suppressed or down regulated in normal cells—plays a major causative role in the onset and progression of human cancer malignancy. For these reasons, the pharmacological antagonism of these cancer specific proteins appears as a very promising target for developing new therapies in oncology.
Pharmacological inhibitors of L1-RT have several effects that indicate that these compounds are capable to counteract androgen-independent mechanism of resistance. Two L1-RT inhibitors, Efavirenz and Nevirapine, have been studied in vitro and in vivo. These molecules are non-nucleoside reverse transcriptase inhibitors (NNRTIs) and have been initially developed for the treatment of HIV infection since they block the reverse transcriptase of the HIV virus and antagonize its replication. Both Nevirapine and Efavirenz are able to inhibit the reverse transcriptase activity of L1-RT in human tumor cell lines (see WO03055493A1).
L1-RT inhibitors can counteract cancer development by three complementary mechanisms: inhibition of proliferation of cancer cell lines (see Sciamanna, 2005; WO03055493A1; WO2006069812A2), induction of cell differentiation and antagonism of epithelial-mesenchymal transition (EMT) (see Sciamanna, 2005; WO03055493A1; WO2006069812A2), and inhibition of intrinsic resistance to apoptosis. Concerning inhibition of intrinsic resistance to apoptosis, L1-RT inhibitors decrease the expression of anti-apoptotic proteins such as Bcl2 and are also very effective in inducing cell death in prostate cancer cell lines that over express proteins of the Bcl2 family such as Bcl-xL that protects them from apoptosis and makes them resistant to treatment.
L1-RT inhibitors thus antagonize tumor progression by acting on multiple cellular mechanisms. These cellular mechanisms play an important role in the pathophysiology of hormone-refractory prostate cancer, and this disease is therefore a natural target for testing the therapeutic effects of L1-RT inhibitors in humans. However, dedifferentiation, epithelial-mesenchymal transition (EMT) and resistance to apoptosis play an important role in pathophysiology of several types of cancers. As a consequence, L1-RT inhibitors can reasonably be expected to be a useful treatment for other cancer pathologies still awaiting treatment and in particular for epithelial cancers (Lee, 2012).
Non-nucleoside RT inhibitors (NNRTIs), such as Efavirenz and nevirapine, have initially been developed as inhibitors of the HIV reverse transcriptase.
Efavirenz (see formula in FIG. 1) is a benzoxazinone and is a particularly well tolerated molecule and used for the treatment of AIDS at the daily dosage of 600 mg/day. Data obtained in humans treated with 600 mg/day of Efavirenz show that plasma concentrations above 1500 ng/ml are observed in approximately 70-80% (Marzolini, 2001) of the subjects.
It has been shown in rats treated with Efavirenz (WO03555493) that tumor progression is inhibited for a dose of approximately 5 mg/kg/day (1 mg/rat/day intraperitoneally, weight of one rat approximately 200 g). In parallel, a pharmacokinetic study in rats published by Balani et al (1999) has shown that an intravenous injection of 5 mg/kg of Efavirenz gives a maximal plasma concentration of the drug of 5 μM, corresponding to 1578 ng/ml.
The daily dosage of 600 mg/day of Efavirenz used for the treatment of AIDS could thus reasonably be expected to be also efficient for the treatment of cancer in humans.
A clinical trial of the use of Efavirenz in the treatment of hormone-refractory prostate cancer was thus launched based on a dose of Efavirenz of 600 mg/day, expecting a response in 70-80% of patients.